Protein folding at extreme temperatures: current issues

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Seminars

in

Cell

&

Developmental

Biology

jo u r n al hom ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / s e m c d b

Review

Protein

folding

at

extreme

temperatures:

Current

issues

Georges

Feller

LaboratoryofBiochemistry,CenterforProteinEngineering-InBioS,UniversityofLiège,InstituteofChemistryB6a,4000Liège-SartTilman,Belgium

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t

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c

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Articlehistory:

Received25February2017

Receivedinrevisedform18August2017

Accepted5September2017

Availableonline25September2017

Keywords: Proteinfolding Extremophiles Triggerfactor Prolylisomerization Thermodynamicstability

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Therangeoftemperaturescompatiblewithlifeiscurrentlyestimatedfrom−25◦_{C,asexemplifiedby} metabolicallyactivebacteriabetweenseaicecrystals,andupto122◦_{Cinhydrothermalventsas} exem-plifiedbythearchaeonMethanopyruskandleri.Inthecontextofproteinfolding,assoonasapolypeptide emergesfromtheribosome,itisexposedtotheeffectsofenvironmentaltemperatures.Recent investi-gationshaveshownthattherateofproteinfoldingisnotadaptedtoextremetemperaturesandshould beveryfastathightemperatureandlowincoldenvironments.Thislackofadaptationisdrivenby kineticconstraintsonproteinstability. Tocounteractthedeleteriouseffectsoffastproteinfolding inhyperthermophiles,chaperonessuchastheTriggerFactorholdandslowdowntherateoffolding intermediates.Prolylisomerization,arate-limitingstepinthefoldingofmanyproteins,isstrongly temperature-dependentandimpairsfoldingofpsychrophilicproteinsinthecold.Thisiscompensatedby reductionoftheprolinecontentincold-adaptedproteins,byanincreasednumberofprolylisomerases encodedinthegenomeofpsychrophilicmicroorganismsandbyoverexpressionofprolylisomerases underlowtemperaturecultivation.Afterfolding,thenativestateisreachedandalthoughextremophilic proteinssharethesamefold,dramaticdifferencesinstabilityhavebeenrecordedbydifferentialscanning calorimetry.

©2017ElsevierLtd.Allrightsreserved.

Contents

1. Introduction...129

2. Thefoldingrateisnotadaptedtoextremetemperatures...130

3. Thetriggerfactorrescuesproteinfoldingathightemperature...131

4. Prolylisomerizationimpairsproteinfoldinginpsychrophiles...132

4.1. Theprolinecontentinproteinsispositivelycorrelatedwithenvironmentaltemperature...132

4.2. Thenumberofprolylisomerasesinextremophilicgenomesiscorrelatedwithtemperature...132

4.3. Prolylisomerasesareoverexpressedinpsychrophiles...133

5. Thermodynamicstabilityofextremophilicproteins...134

5.1. Microcalorimetricstudies...134 5.2. Stabilitycurves...134 6. Conclusions...135 Acknowledgments...135 References...135 1. Introduction

Inrecentyears,microbiologicalinvestigationsofenvironments previously regarded asabiotic have considerably expandedthe

Abbreviations: TF,triggerfactor;PPIase,prolylisomeraseorpeptidyl-prolyl

cis/transisomerase; GFP,green fluorescentprotein; DSC,differentialscanning

calorimetry.

E-mailaddress:gfeller@ulg.ac.be

spectrum of biological temperatures. Metabolically active bac-teria have been found at −20◦_{C, thriving in the liquid brine}

veinsbetweenseaicecrystals[1,2].Morerecently,thebacterium Planococcus halocryophilus isolated from Arctic permafrost was found togrowand todivide at −15◦_{C and to displayresidual}

metabolic activityat −25◦_{C [3], which possibly represents the}

lowertemperaturelimitbeforedormancy.Theselowtemperature extremophiles are termed psychrophiles. At the other extrem-ityofthetemperaturerangecompatiblewithlife,thermophiles areknownfordecadesbuthyperthermophiles havepushedthe https://doi.org/10.1016/j.semcdb.2017.09.003

playa pivotalrole astheydrivethecellcycle andmetabolism. Atlowtemperature,enzymeshavetoremainactiveandproteins havetomaintainadequatefunctionaldynamicsattemperatures thatslowdownmolecularmotions.Thisisreachedbyadopting amobileandflexiblestructurethroughreductionofalltypesof weakinteractionsinvolvedintheshapingofproteinconformation suchashydrogenbonds,saltbridges,vanderWaalscontacts,helix dipole,etc.andthehydrophobiceffect.Butthepricetopayforsuch animprovedstructuraldynamicsisthepronouncedheat-labilityof psychrophilicproteins[8–12].Incontrast,thermophilicand hyper-thermophilicproteinshavetomaintainafunctionalnativestate atelevatedtemperaturesthatwouldotherwiseunfoldmesophilic proteins.Their robustand heat-stablestructure arisesfromthe improvementinstrengthandnumberofalltypesofweak inter-actionsandstructuralfactorsstabilizingtheproteinconformation [13–15].

Here,wewillfocusonproteinfolding,acrucialtopicforthe biophysicalunderstandingoflifeatextremetemperatures.Indeed, assoonasanascentpolypeptideemergesfromtheribosome,it isexposedtotheeffectsofenvironmentaltemperatures.Recent investigationshaveaddressedsomeessentialquestions:i)whatis theeffectofextremeenvironmentaltemperaturesontheprotein foldingrate;ii)theTriggerFactor(TF)isthefirstchaperone interact-ingwithnascentchains:howdoesithelpproteinfoldingatextreme temperatures;iii)howdoprolylisomerasescatalyzeprolyl isomer-ization,arate-limitingstepinproteinfoldingandiv)whatarethe propertiesofthefinalnativestateofproteinadaptedtothese tem-peratures?Manyofthereviewedresultshavebeenobtainedwith TFasamodelprotein.InBacteria,TFisaribosome-bound chaper-oneinteractingwithvirtuallyallsynthesizedpolypeptidesandit alsopossessesapeptidyl-prolylcis/transisomerase(PPiase) activ-ity[16].Thischaperonehasbeenisolatedfrommodel bacteria: theAntarcticpsychrophilePseudoalteromonashaloplanktis,afast growingstrainatlowtemperature[17],themesophilicreference EscherichiacoliandthehyperthermophileThermotogamaritima[4].

2. Thefoldingrateisnotadaptedtoextremetemperatures Inordertodeterminethefoldingrateconstant,theunfolded protein(usuallyinureaorguanidiniumchloride)isdilutedwitha bufferanditsrefoldingismonitoredbyastopped-flow spectropho-tometerastheprocessiscompletedwithinafewseconds.Similar experimentsareperformedforunfolding,startingwiththenative state(Fig.1).

Byusingarangeoffinaldenaturantconcentrations,a“Chevron plot”isconstructed(seeFig.2asanexample).

Theleft armof theplot correspondsto foldingkinetics and therightarmdepictsunfoldingkinetics.Finally,themostrelevant parameters,themicroscopicrateconstantsforfoldingk_f(H2O)and

forunfoldingk_u(H2O)areobtainedbyextrapolationontheYaxis,

i.e.intheabsenceofdenaturant,inH2O.

Fig. 1.Representative folding and unfolding fluorescence traces recorded by

stopped-flow.Rateconstantsareobtainedbyadjustingoneorseveralexponential

functionstothekinetictraces.Unpublisheddatafrom[18]usingapsychrophilicTF.

Fig.2. Chevronplotsoftherateconstantskappforunfolding(closedsymbols,right

arms)andrefolding(opensymbols,leftarms)oftryptophansynthase␣subunit

fromthepsychrophileS.frigidimarina(,)andfromthemesophileE.coli(䊉,)at

25◦C.

Reprintedfrom[19]bypermissionofOxfordUniversityPress.

Becausetheserateconstantsstronglydifferinproteinsandare alsoaffected bytheexperimental setup,it isessential to com-pareseriesofhomologousextremophilicproteinsunderthesame experimentalconditions[20].Thishasbeenperformedusingthe above-mentionedTF[18].Forthesakeofclarity,Fig.3isa simpli-fiedversionoftheoriginalwork(onlythemainphasesaredepicted) thatallowsonetodrawseveralconclusions.Asfarasfoldingis con-cerned,atthesametemperature,thepsychrophilicTF(Fig.3,left panel,blueline)foldsslowerthanitsmesophilichomologue(black line)and thehyperthermophilicprotein(redline) foldsslightly faster.Inthenarrowwindowoftemperatureaccessibleto stopped-flowexperiments,thetemperaturedependenceofthisfoldingrate wassimilarfor thethree proteins.Accordingly,onehasto con-cludethatthefoldingrateisnotadaptedtoextremetemperatures becauseunderenvironmentalconditionsthehyperthermophilic proteinshouldfoldextremelyfast,whereasthepsychrophilic pro-teinshouldfoldevenslowerinthecold.Thesameobservationwas madeforapsychrophilictryptophansynthasesubunit(Fig.2)as comparedwithitsE.colihomologue[19]andsuggestsacommon behaviorofcold-adaptedproteins.Ontheotherhand,proteinsfrom

Fig.3. Close-upofordinateextrapolationsfromChevron-plots.Dataareshownfor

psychrophilic(blue),mesophilic(black)andhyperthermophilic(red)TFsat9◦_C.

Extrapolationsoftherateconstantsforthedeterminationofthemicroscopicrate

constantsforfoldingkf(H2O)andunfoldingku(H2O)intheabsenceofdenaturant.

Adaptedfrom[18].

thermophilesandhyperthermophilesgenerallyfoldslightlyfaster [21–23]orwithsimilarrateconstants[21,24–26]ascomparedwith theirmesophilichomologues.

In contrastto folding, thedifferences in theunfolding rates of extremophilic TF are much larger (Fig. 3, right panel). The hyperthermophilicproteinunfoldsveryslowly,whereasthe psy-chrophilicproteinunfoldsfasterascomparedwiththemesophilic counterpart. In fact, the slow unfolding of hyperthermophilic proteinshasbeenpreviouslyidentifiedasthemainkinetic con-tributiontotheunusuallyhighstabilityoftheseproteins[24–29]. Indeed,inasimpletwo-stateequilibriumbetweentheunfolded stateUandthenativestateNshowninEq.(1),

Ukf

ku

N (1)

aforwarddisplacementoftheequilibriumtowardsN,i.e.an increaseofstability,isobtainedbyaslowerunfoldingrateku,as observedinheat-stableproteins.Conversely,abackward displace-mentoftheequilibrium,i.e.adecreaseofstability,isreachedby afasterunfolding ratek_u andthisisprecisely whatisobserved for thenatively unstable psychrophilicproteins (Figs. 2 and 3) [18,19]andalsopredictedbymoleculardynamicssimulations[30]. Verysignificantly,stabilizationofapsychrophilicalpha-amylase bysite-directedmutagenesisresultsinaslowerunfoldingrateof themutants[31],underliningthekineticstrategyleadingtothe nativelyunstableconformationofpsychrophilicproteins. There-fore,thereisafinetuningoftheunfolding ratevalues inorder toadjustproteinstabilitytotheenvironmentaltemperature.This resultsinacontinuumfromfasttoslowunfoldingratesinunstable tohyperstableproteins.

However,thesamerationalecanbeappliedtothefoldingrate k_f.InEq.(1),anincreaseinstabilityisobtainedbyafastfolding ratek_f,whereasa lowerstability isreachedbya slowerfolding rate.Again,thisisexactlywhatisobservedforhyperthermophilic and psychrophilicproteins, respectively (Figs. 2 and 3). Signifi-cantlyalso,allmutantsofahyperthermophilicenzymedestabilized

Fig.4. GFPrefoldingassistedbytriggerfactors.Dataareshownforpsychrophilic

(blue),mesophilic(black)andhyperthermophilic(red)TFs.Refoldingtimecourses

ofacid-denaturedGFPat15◦_{Crecordedbyfluorescence.Fluorescenceintensity}

(extrapolatedtotheinfinite)ofspontaneouslyrefoldedGFP(noTF)istakenas100%.

Adaptedfrom[33].

bysite-directedmutagenesisdisplayedbothfasterunfoldingrates and slowerfolding rates than thewild-type protein [25,32]. In summary, the nativelyunstable structure ofpsychrophilic pro-teinsisgainedviabothafastunfoldingrateandaslowfolding rate.Conversely,hyperthermophilicproteinsarecharacterizedby aslowunfoldingrateand,toalowerextent,byafastorunchanged foldingrate.Itfollowsthatthefoldingratecannotbeadaptedto extremetemperaturesbecauseadjustmentsofproteinstabilityto environmentaltemperatureisunderkineticcontrol.Loweringthe foldingrateinhyperthermophileswouldresultinprotein desta-bilization,whereasacceleratingthefoldingrateinpsychrophiles wouldincreaseproteinstability.

What are the physiological consequences for the nascent polypeptide?Intuitively,theslowfoldingrateofpsychrophilic pro-teinsshouldnotbeaconcern,exceptthatitcontributestoslow downthecellcycle,asobservedintheenvironmentbutresulting frommany othertemperature sensitivecellularevents. In con-trast,boththefastfoldingrateofhyperthermophilicproteinsand hightemperatureshouldhavedeleteriouseffectsforthenascent polypeptidebecausetheyincrease theprobabilityofmisfolding eventsandaggregationofmisfoldedspecies.Thisaspectisanalyzed inthenextsection.

3. Thetriggerfactorrescuesproteinfoldingathigh temperature

Ifthefoldingrateisnotadaptedtoextremetemperaturesinthe testtube,chaperonescanhaveapivotalroletorescueprotein fold-inginvivo.TFisthefirstfoldingassistantactingco-translationally onsynthesizedpolypeptidesinbacteria.Thisisanobvious candi-datetotesttheeffectsofchaperonesandthishasbeenperformed usingtheabove-mentionedextremophilicTFs[33].Fig.4illustrates a classical refoldingexperiment in whichaciddenatured green fluorescentprotein(GFP)isallowedtorefoldafterbuffer neutral-ization,eitheraloneorinthepresenceofaddedTF.Thefluorescence intensityofGFP isdirectlyproportional totheconcentrationof nativeGFPin theexperiment.ThemesophilicTF fromE.coliis averyefficientfoldasethatcanimprovetheyieldofnativeGFP upto150%.ThepsychrophilicTFisalsoafoldasewithhowevera weakerchaperoneactivity.Thiscanbetentativelyrelatedtothefact thatlowtemperatureslowsdownproteinfoldingandreducesthe probabilityofmisfoldingandaggregation.Asamatteroffact,low

Fig.5. GFPrefoldinginthepresenceofTFfromthehyperthermophileT.maritima

andGroELSat15◦_{C.FluorescencetimecoursesofGFPaloneandinthepresenceof}

GroELS,TmTForTmTF+GroELS.Inthesequentialexperiment,additionofGroELS

after300sisindicatedbyanarrow.

Adaptedfrom[33].

Fig.6.Transandcisisomersofapeptidyl-prolylpeptidebond.

Reprintedwithpermissionfrom[41].Copyright2009AmericanChemicalSociety.

temperaturecultivationofE.colifrequentlyavoidstheformationof insolubleinclusionbodiesofrecombinantproteins[34],aswellas expressioninapsychrophilichostatlowtemperature[35]. Further-more,lowtemperatureweakensthehydrophobiceffect.Therefore, anefficientfoldaseispossiblynotrequiredbypsychrophilesincold conditions.Butthemostinsightfulresultwasobtainedwiththe hyperthermophilicTF.AsshowninFig.4,TFfromT.maritimais notafoldase(itdoesnotpromotethespontaneousGFPrefolding) butinsteaditisaholdase:itbindstotherefoldingGFPand dras-ticallydecreasesitsfoldingrate.Accordingly,theholdasefunction ofthisTFshouldberegardedasthemainadaptationin hyperther-mophilicbacteriainordertocounteractthedeleteriouseffectsof hightemperatureonproteinfolding.

Interestingly,whenthewell-knownGroELSchaperoneisadded to the refolding mixture, a burst in refolded GFP is observed (Fig.5).Asimilarresulthasbeenreportedfor thethermophilic TF fromThermus thermophilus[36].This strongly suggests that thehyperthermophilicTF actsasa carrieroffolding intermedi-atesfordeliverytodownstreamchaperonesandfinalmaturation. Furthermore,thestronglyimprovedyieldofnativeGFPinthe pres-enceofbothTFandGroELSsuggeststhatthehyperthermophilicTF maintainsintermediatespeciesinafolding-competentstatewhich favorstheactionofdownstreamchaperones.

To explore the holdase function, ANS titration and isother-maltitrationcalorimetryhaverevealedahydrophobicchaperone cavitywhichpotentiallybindsapolarcomponentsoffolding inter-mediates, therefore lowering their rate of internalization and consequently the folding rate of the client protein [33]. The stoichiometryofinteractionwith␣-casein,anintrinsically disor-deredprotein(nativelyunfolded),indicatedahighernumber of

proteins[39].Thisarisesfromtheweakstericconstraintsexerted bythepyrrolidineringofprolinetofavoreitherthecisorthetrans conformationintheunfoldedstateandtotheslowisomerization involvingtherotationaboutthepeptidebond,whichhasapartial double-bondcharacter(Fig.6).Itfollowsthatthetimespentby foldingintermediatestoexploretheconformationalspaceandto adopttherequiredprolylcisortransconformationislongerthan forotheraminoacidsidechainswhicharealmostinvariably con-strainedintrans.Prolylisomerizationintrinsicallypossessesahigh activationenergyandisthereforestronglytemperaturedependent [40].Inthecontextofextremetemperatures,onecanintuitively assumethatprolylisomerizationisveryfastinhyperthermophiles andshouldnotbeaconcernforproteinfolding.Incontrast,slow prolylisomerizationatlowtemperatureshouldimpairthefolding ofpsychrophilicproteins.Severallinesofevidenceindicatethat thisisindeedtheactualsituation.

4.1. Theprolinecontentinproteinsispositivelycorrelatedwith environmentaltemperature

The first observation refers tothe proline content which is low in psychrophilicproteinsand high in heat-stable polypep-tides.Forinstance,inhomologousalpha-amylases(∼50kDa)the psychrophilicenzymecontains13prolines,themesophilic homo-loguehas19prolinesandtheheat-stableenzymehas25prolines, i.e.nearly twicethecontentof thecold-adaptedprotein.These differenceshavebeenrelatedtoadjustmentsofproteinstability [8,10,11].Indeed,intheprolineresidue,thestructureofthe pyrro-lidineringbondedtothemainchainnitrogenlocksadihedralangle andseverelyrestrictsrotationsofthebackbone.Thislocal rigid-ityinducedbytheprolylresidueisawell-knownstructuralfactor improvingproteinstability[14,15,42].Consequently,heat-stable proteinsdisplayahighprolinecontent,whereaspsychrophilic pro-teinstendtodecrease thiscontent inorder toreacha natively unstableconformation.Inthecontextofproteinfolding,thehigh prolinecontentofhyperthermophilicproteinsshouldnotbea con-cernasaresultoffastprolylisomerizationathightemperature, whereasthelowprolinecontentofpsychrophilicproteinsreduces theprobabilityofslowandrate-limitingfoldingsteps.Therefore, thelowprolinecontentinpsychrophilicpolypeptideshastwo dis-tinctcontributions:itavoidsprolyl-limitedfoldingeventsandit destabilizesthenativestate.

4.2. Thenumberofprolylisomerasesinextremophilicgenomesis correlatedwithtemperature

Cellsareequippedwithspecialized catalysts,peptidyl-prolyl cis/transisomerases(PPiasesorrotamases),whichspeed-upprolyl isomerization in proteins. Fig. 7 illustrates a classical exper-iment in which increasing amounts of PPiase proportionally accelerate the folding of a proline-limited protein, revealing a trueenzymatic activity[43].The second observationindicating

Fig.7. CatalyticactivityofPPiasesonproteinfolding.Therefoldingrateofamodel

protein(RCM-T1,reducedandcarboxymethylatedribonucleaseT1)limitedby

pro-lineisomerizationcanbeslow(1).AdditionofincreasedamountsofPPiase(2–7)

acceleratesthefoldingrateofthemodelprotein,asshownbytheincreasesof

fluorescence.

Reprintedfrom[43]bypermissionfromMacmillanPublishers:TheEMBOJournal.

Copyright1997.

Table1

Prolylisomerasesencodedinthegenomeofrepresentativeextremophilicand

mesophilicGram-negativebacteriaandtheirenvironmentaltemperatures.

Strain Temperature PPiasegenes Colwelliapsychrerythraea34H <0◦_{C 18}

PseudolateromonashaloplanktisTAC125 <0◦_{C 15}

Pseudomonasextremaustralis14-3 <0◦_{C 14}

EscherichiacoliK12 37◦_{C 10}

ThermusthermophilusHB8/HB27 65◦_{C 4}

ThermotogamaritimaMSB8 85–90◦C 1 AquifexaeolicusVF5 85–95◦C 1

that prolyl isomerization is influenced by extreme

tempera-turesisthenumberofgenesencoding PPiasesinextremophilic

genomes. Various types of PPiases are expressed in cells

(FKBP-type, cyclophilin-type, parvulin-type) and all have been

screened in representative extremophilic genomes using MaGe

(www.genoscope.cns.fr/agc/microscope/home/index.php).

Table1 shows that when compared withE. coli, the Gram-negativemesophilicreference,psychrophilicgenomes containa highernumberofPPiasegenes,nearlytwiceforC.psychrerythrae. Conversely,whenthegrowthtemperatureincreases,thenumber ofPPiasesgraduallydropsto4genesat 65◦C andto1 geneat 85–95◦C.Gram-positivebacteriaencodealowernumberofPPiases butthesametrendisobserved.ThestrainsPlanococcusantarcticus andP.halocryophilusthrivingaround0◦Cencode7and6PPiases, respectively,whereasthemesophilesB.subtilisandEnterococcus speciesallcontain4PPiases.IncontrastGeobacillusand Thermin-colaspeciesthrivingat60◦Conlycontain3PPiases.Butthemost convincingevidencethatthePPiasegenomiccontentiscorrelated totemperatureisprovidedbymethanogenicarchaeabecausethey havecolonizedthelargestrangeofenvironmentaltemperatures.As showninTable2,mesophilicarchaeaencode4–5PPiases,whereas inthe65–85◦Crangeonly2PPiasesarefound.Significantly,M. kandleri,themost heat-resistantorganismknownto date,only encodesonePPiase.Inaddition,thegenomeofmost hyperther-mophilicarchaeasuchasPyrococcusfuriosus,Sulfolobussolfataricus orThermococcusspeciesalsoonlyencodesonePPiase.

Overall, this analysis is a strong indication that prolyl iso-merization is sufficiently fast at high temperature, whereas in psychrophilesthisisomerizationrequirespowerfulcatalytic assis-tancebyafullsetofPPiases.Furthermore,thepersistenceofonly onePPiaseinhyperthermophilicarchaeasuggeststhatthefolding ofa subset ofessential proteinsis limitedby prolyl isomeriza-tion. Alternatively, other functionsof archaeal PPiases, suchas

Table2

Prolylisomerasesencodedinthegenomeofrepresentativeextremophilicand

mesophilicmethanogenicarchaeaandtheirenvironmentaltemperatures.

Strain Temperature PPiasegenes Methanosarcinabarkeri 25◦_{C 5} Methanosarcinaacetivorans 37◦_{C 5} Methanosarcinamazei 37◦_{C 5} Methanococcusmaripaludis 37◦_{C 4} Methanococcusaeolicus 45–50◦C 3 Methanothermobacterthermautotrophicus 65–70◦C 2 Methanocaldococcusjannaschii 85◦_{C 2} Methanopyruskandleri 122◦_{C 1}

Fig.8. CatalysisofrefoldingofRCM-T1(reducedandcarboxymethylated ribonu-cleaseT1).DataforthepsychrophilicTF(,blue),E.coliTF(䊉,black)andthe hyperthermophilicTF(䊏,red).Theapparentrateconstantofprolylisomerization duringrefoldingofRCM-T1isplottedasfunctionoftheTFconcentration. Adaptedfrom[33].

chaperoneoranti-aggregationactivities,couldberesponsiblefor theirgenomicpersistence[44].Itshouldbementioned thatthe aboveanalysisdepictsageneraltrendbutnotanabsoluteruleas exceptionsoccurinthedatabase.Thisanalysisstronglyrelieson thequalityofgenomeannotationsandotherfactors,suchasthe microorganismlifestyle(salinity,pressure,ecologicalniche...),are possiblyinvolved.

4.3. Prolylisomerasesareoverexpressedinpsychrophiles

Enzymeactivitydisplaystypicaladaptivetraitsto environmen-taltemperature[8,45] andit wasthereforeofinterest tocheck PPiasesactivityinextremophiles.ThereisonlyonePPiaseshared bymodelextremophilicbacteriaandthisistheTFagain.The PPi-aseactivitywasanalyzedusingnewlydevelopedsubstratesand methodologies[33].Fig.8depictssuchanexperiment inwhich theincreaseofthefoldingrateofaproline-limitedprotein sub-strateisplottedasafunctionofthePPiaseconcentrationinorder tocalculatethe catalyticefficiency k_cat/K_m ofthe isomerization reaction.ItcanbeseenthatthePPiaseactivityofthe hyperther-mophilicproteinisextremelylowandclosetothedetectionlimit. Thiswasexpected becausehyperthermophilicenzymes require hightemperaturetobecomefullyactivatedandtheirrigid struc-tureprecludessignificantactivity,evenatroomtemperature.More surprisingwasthesamePPiaseactivitysharedbyboththe psy-chrophilicandmesophilicTFs.Indeed,inmostcasestheactivity of psychrophilic enzymes is very high in order to compensate for the decreaseof reaction rates inherent tolow temperature [10–12].ThepsychrophilicPPiaseescapesthisrulebutthereasons

Fig.9. Thermalunfoldingofextremophilicproteins.ThermogramsofDNA-ligases

recordedbydifferentialscanningmicrocalorimetryshowing,fromlefttoright,

psy-chrophilic(blue),mesophilic(black)andhyperthermophilic(red)proteins.

Adaptedfrom[52].

remainunclear.Thiscanbetentativelyrelatedtothepeculiarbut stillhypotheticalreactionmechanismofPPiases[46]which possi-blyprecludescoldadaptation.However,proteomicstudieshave broughtanunsuspectedanswertothis paradox[47].Indeed, it wasfoundthatthis TFisoverexpressed nearly40timesbythe psychrophile P.haloplanktis under low temperaturecultivation. Apparently,ifthePPiasespecificactivitycannotbeimprovedatlow temperature,themicroorganismadaptsbydramaticallyincreasing theenzymeconcentrationandthereforetheavailablecellular PPi-aseactivity.Moreover,overexpressionofPPiaseshasbeenreported inalmostallproteomicstudiesofpsychrophilessofar[48]andthis isprobablythemostfirmlyestablishedresultsharedbyproteomics ofpsychrophiles.Again,thispointstothenegativeeffectof pro-lylisomerizationonfoldingofpsychrophilicproteinsandtothe requirementofcatalyticassistancebyPPiases.

5. Thermodynamicstabilityofextremophilicproteins

Afterallfoldingevents,thenativestateisreachedandalthough extremophilicproteinssharethesamefoldand3Dstructures,they display dramatic differences in terms of stability. The energet-icsofstructurestability wasessentiallyanalyzedbydifferential scanningcalorimetry(DSC)ofhomologousextremophilicproteins [18,45,49–53].

5.1. Microcalorimetricstudies

Ademonstrativeexampleofmicrocalorimetricrecordsforthe heat-inducedunfoldingofextremophilicproteinsisillustratedin Fig.9.Theseproteinsclearlyshowdistinctstabilitypatternsthat evolvefromasimpleunfoldingprofileintheunstablepsychrophilic proteintoamorecomplexprofileinthestablehyperthermophilic counterpart.Theunfoldingofthecold-adaptedproteinoccursat muchlowertemperaturesasindicatedbythetemperatureof half-denaturationT_m=33◦C,givenbythetopofthetransition(Table3). Accordingly,this proteinspontaneouslyunfoldsat amesophilic temperatureof37◦C.Bycontrast,thehyperthermophilicprotein unfoldsaround 100◦C. Melting point values up to 150◦C have beenreportedforhyperthermophilicarchaealproteins[54,55].The calorimetricenthalpy_Hcal(areaunderthecurvesinFig.9) cor-respondstothetotalamountofheatabsorbedduringunfolding, butitalsoreflectstheenthalpyofdisruptionofbondsinvolvedin

ingtoacooperative,all-or-noneprocess,revealingauniformlylow stabilityofitsarchitecture.Bycontrast,bothhomologousproteins displaytwotothreetransitions(indicatedbydeconvolutionofthe heatcapacityfunctionin Fig.9).Therefore, theconformationof thesemesophilicandhyperthermophilicproteinscontains struc-turalblocksorunitsofdistinctstabilitythatunfoldindependently. Fromtheseobservations,itcanbeconcludedthatpsychrophilic proteinspossessafragilemolecularedificethatisuniformly unsta-bleand stabilizedbyfewerweakinteractionsthanhomologous mesophilicproteins.Bycontrast,hyperthermophilicproteinsare robustmolecules,madeofvariousstabilitydomainsandstabilized byahighnumberofenthalpy-drivenweakinteractions.

5.2. Stabilitycurves

Thethermodynamicstabilityofaproteinthatunfoldsreversibly accordingto a two-state mechanism (between thenativestate NandtheunfoldedstateU)isdescribedbytheclassical Gibbs-Helmholtzrelation:

GN-U = HN-U−TSN-U (2)

Thelatterrelationcanberewrittenforanytemperature(T)using theparametersdeterminedexperimentallybyDSC:

GN-U(T) = Hcal(1-T/Tm)+Cp(T-Tm)-TCpln(T/Tm) (3)

whereCpisthedifferenceinheatcapacitybetweenthenative andtheunfoldedstate.ComputingEq.(3)inatemperaturerange wherethenativestateprevailsinsolutionprovidestheprotein stability curve[56],i.e. the free energyof unfolding as a func-tion of temperature (Fig. 10). In other words,this is thework requiredtodisruptthenativestateatanygiventemperature[57] andisalsoreferredtoasthethermodynamicstability.Bydefinition, GN-UiszeroatTm,atequilibrium.AttemperaturesbelowTm,the stabilityincreases,asexpected,butperhapssurprisinglyforthe non-specialist,thestabilityreachesamaximumthenitdecreases atlowertemperatures(Fig.10).Infact,thisfunctionpredictsa tem-peratureofcold-unfolding,whichisgenerallynotobservedbecause itoccursbelow 0◦C [58].Increasingthestabilityofa proteinis essentiallyobtainedbyliftingthecurvetowardshigherfreeenergy values[59,60],asexemplifiedbythehyperthermophilicprotein (Fig.10),whereasthelowstability ofapsychrophilicproteinis reachedbyaglobalcollapseofitscurve.Inallcases,thereisno sig-nificantshiftofthecurvestowardshighorlowtemperatures.Asfar asextremophilesareconcerned,oneofthemostpuzzling obser-vationsisthatmostproteinsobeythispattern,i.e.whateverthe microbialsource,eitherfromhotspringsorfromseaice,the maxi-malstabilityoftheirproteinsisclusteredaroundroomtemperature [59,61,62].Thisindicatesthatthevariousandsometimesopposites forcesdrivingfoldingareoptimallybalancedinthistemperature range.Itshouldbenotedthatwhenstabilitycurvesarecomputed fromequilibriumunfoldingbychemicaldenaturants,shiftsofthe temperatureformaximalstabilitytowardshigherorlower

temper-Fig.10.Gibbsfreeenergyofunfolding,orconformationalstability,ofhomologous

proteins.Theworkrequiredtodisruptthenativestateisplottedasafunctionof

temperatureforpsychrophilic(blue,PhTF),mesophilic(black,EcTF)and

hyperther-mophilic(red,TmTF)proteins.

Adaptedfrom[18].

atureshavebeenreported[24,63].Suchdiscrepanciesremaintobe clarifiedbutpossiblyoriginatefromthedistinctunfolding mech-anismsandthenatureoftheunfoldedstates.TheCpparameter inEq.3contributestothecurvatureofthestabilitycurveanda decreaseofthisvalueinducesaflatteningofthefunction. Reduc-tionoftheCpvaluehasbeenreportedforsomethermophilicand hyperthermophilicproteins[18,23,64–66].Ithasbeenshownthat thedifferenceinheatcapacitybetweenthenativeandunfolded statesdecreaseswithtemperatureandvanishesataround120◦C formostmesophilicproteins[67].Itfollowsthatthehigherthe meltingtemperature,thelowertheCpvalue.

Accordingtothebell-shapedstabilitycurve,the environmen-taltemperaturesformesophilesandhyperthermophileslieonthe rightlimbofthecurveandobviously,thethermaldissipativeforce isusedtopromotemolecularmotionsinthesemolecules.By con-trast,theenvironmentaltemperaturesforpsychrophileslieonthe leftlimbofthestabilitycurve.Itfollowsthatmolecularmotions inproteinsatlowtemperaturesaregainedfromthefactors ulti-matelyleadingtocold-unfolding[49],i.e.thehydrationofpolar andnon-polargroupsandtheweakeningofthehydrophobiceffect [68].Theoriginofflexibilityinpsychrophilicproteinsatlow tem-peraturesis therefore drasticallydifferentfrommesophilic and hyperthermophilicproteins,thelattertakingadvantageofthe con-formationalentropyrisewithtemperaturetogaininmobility.

Asurprisingconsequenceofthefreeenergyfunctionforthe psychrophilicproteinshowninFig.10isitsweakstabilityatlow temperatureswhencomparedwithmesophilicandthermophilic proteins, whereasit wasintuitively expectedthat cold-adapted proteinsshouldalsobecoldstable.Thisproteinisinfactbothheat andcoldlabile.Asaresult,coldunfoldingofpsychrophilicproteins hasbeenexperimentallyrecordedatthetemperaturepredictedby thestabilitycurve[18,49],providingvalidationofthefreeenergy function.

6. Conclusions

Theliteratureonproteinfoldingintemperatureextremophiles is still scarcebut this shouldimprove rapidlybecause, besides fundamentalaspects,thetopicissignificantlyrelevantin biotech-nology,suchasfortheexpressionofsolublerecombinantproteins atlowtemperaturebypsychrophiles[35]orforthesynthesisof

robustenzymecatalystsusedinharshindustrialconditions[69]. The topicis alsoof interest for astrobiologyas lifemight have evolvedoncoldorhotplanets[70].Theavailableresultsunderline thenecessityofinvestigatingseriesofhomologousextremophilic andmesophilicproteinsinordertoscreenthelargestspectrum of biological temperatures. Current studiesusing extremophilic proteinswhichunfoldfullyreversiblyaccordingtoaperfect two-statemechanismshouldrefinetheavailabledata.Multidisciplinary approachesarealsoneededbecause,forinstance,coldadaptation isnotalwaystheconverseofhotadaptation.Inaddition,multiple factorscanbeinvolvedasillustratedherebychaperonesorbythe effectofprolylisomerization.

Finally, the main drawback in protein folding studies of extremophilesisthefactthattherelatedexperimentscannotbe performed at theenvironmentaltemperatures. Atlow temper-atures, condensation on optics strongly perturbs spectroscopic signals and requires abundant nitrogen flushing, which is not alwaystechnicallyfeasible.Athightemperatures,foldingevents becomesofastthattheycannotberecordedandallmodelprotein substratesusedintheseexperimentsaremesophilic,suchasGFP, andunfoldoraggregateattemperatureswellbelowthose encoun-teredbythermophiles.Inallcases,resultsobtainedinalimited windowoftemperatures, aswellastheassociated values,have tobeextrapolatedtoeitherloworhightemperatures,whichcan becontroversial.Reviewersaresometimesreluctanttotakethese limitationsintoaccount.

Acknowledgments

I gratefully thank AndréMatagne (Liège University), Philipp Schmidpeter and Franz Schmid (Bayreuth University) for their expertscientificinputinsomeofourworkscitedhere.Ialsothank S.D’Amicoforhisearliercontribution,aswellaspreviousPhD stu-dents,F.Piette,C.Struvay,A.CipollaandA.GodinduringtheirFRIA fellowship.Worksattheauthor’slaboratoryweresupportedbythe F.R.S-FNRS,Belgium(FondsdelaRechercheFondamentaleet Col-lective,contractnumbers2.4535.08,2.4523.11andU.N009.13)and bytheBelgianprogramofInteruniversityAttractionPoles(iPros P7/44)initiatedbytheFederalOfficeforScientific,Technicaland CulturalAffairs.

References

[1]J.W.Deming,Psychrophilesandpolarregions,Curr.Opin.Microbiol.5(2002) 301–309,S1369527402003296.

[2]K.Junge,H.Eicken,J.W.Deming,BacterialActivityat−2to−20degreesCin

Arcticwintertimeseaice,Appl.Environ.Microbiol.70(2004)550–557,http://

dx.doi.org/10.1128/AEM.70.1.550-557.2004.

[3]N.C.Mykytczuk,S.J.Foote,C.R.Omelon,G.Southam,C.W.Greer,L.G.Whyte,

Bacterialgrowthat−15degreesC;molecularinsightsfromthepermafrost

bacteriumPlanococcushalocryophilusOr1,ISMEJ.7(2013)1211–1226,http://

dx.doi.org/10.1038/ismej.2013.8.

[4]H.Latif,J.A.Lerman,V.A.Portnoy,Y.Tarasova,H.Nagarajan,A.C.

Schrimpe-Rutledge,R.D.Smith,J.N.Adkins,D.H.Lee,Y.Qiu,K.Zengler,The

genomeorganizationofThermotogamaritimareflectsitslifestyle,PLoSGenet.

9(2013)e1003485,http://dx.doi.org/10.1371/journal.pgen.1003485.

[5]G.Deckert,P.V.Warren,T.Gaasterland,W.G.Young,A.L.Lenox,D.E.Graham,

R.Overbeek,M.A.Snead,M.Keller,M.Aujay,R.Huber,R.A.Feldman,J.M.

Short,G.J.Olsen,R.V.Swanson,Thecompletegenomeofthe

hyperthermophilicbacteriumAquifexaeolicus,Nature392(1998)353–358,

http://dx.doi.org/10.1038/32831.

[6]E.Blochl,R.Rachel,S.Burggraf,D.Hafenbradl,H.W.Jannasch,K.O.Stetter,

Pyrolobusfumarii,gen.andsp.nov.,representsanovelgroupofarchaea,

extendingtheuppertemperaturelimitforlifeto113degreesC,

Extremophiles1(1997)14–21,http://dx.doi.org/10.1007/s007920050010.

[7]K.Takai,K.Nakamura,T.Toki,U.Tsunogai,M.Miyazaki,J.Miyazaki,H.

Hirayama,S.Nakagawa,T.Nunoura,K.Horikoshi,Cellproliferationat122

degreesCandisotopicallyheavyCH4productionbyahyperthermophilic

methanogenunderhigh-pressurecultivation,Proc.Natl.Acad.Sci.U.S.A.105

[15]C.Vieille,G.J.Zeikus,Hyperthermophilicenzymes:sources,uses,and

molecularmechanismsforthermostability,Microbiol.Mol.Biol.Rev.65

(2001)1–43,http://dx.doi.org/10.1128/MMBR.65.1.1-43.2001.

[16]A.Hoffmann,B.Bukau,G.Kramer,Structureandfunctionofthemolecular

chaperoneTriggerFactor,Biochim.Biophys.Acta2010(1803)650–661,

http://dx.doi.org/10.1016/j.bbamcr.2010.01.017.

[17]C.Medigue,E.Krin,G.Pascal,V.Barbe,A.Bernsel,P.N.Bertin,F.Cheung,S.

Cruveiller,S.D’Amico,A.Duilio,G.Fang,G.Feller,C.Ho,S.Mangenot,G.

Marino,J.Nilsson,E.Parrilli,E.P.Rocha,Z.Rouy,A.Sekowska,M.L.Tutino,D.

Vallenet,G.vonHeijne,A.Danchin,Copingwithcold:thegenomeofthe

versatilemarineAntarcticabacteriumPseudoalteromonashaloplanktis

TAC125,GenomeRes.15(2005)1325–1335,http://dx.doi.org/10.1101/gr.

4126905.

[18]C.Struvay,S.Negro,A.Matagne,G.Feller,Energeticsofproteinstabilityat

extremeenvironmentaltemperaturesinbacterialtriggerfactors,

Biochemistry52(2013)2982–2990,http://dx.doi.org/10.1021/bi4002387.

[19]D.Mitsuya,S.Tanaka,H.Matsumura,N.Urano,K.Takano,K.Ogasahara,M.

Takehira,K.Yutani,M.Ishida,Strategyforcoldadaptationofthetryptophan

synthasealphasubunitfromthepsychrophileShewanellafrigidimarina

K14-2:crystalstructureandphysicochemicalproperties,J.Biochem.155

(2014)73–82,http://dx.doi.org/10.1093/jb/mvt098.

[20]A.A.Nickson,J.Clarke,Whatlessonscanbelearnedfromstudyingthefolding

ofhomologousproteins?Methods52(2010)38–50,http://dx.doi.org/10.

1016/j.ymeth.2010.06.003.

[21]D.Perl,C.Welker,T.Schindler,K.Schroder,M.A.Marahiel,R.Jaenicke,F.X. Schmid,Conservationofrapidtwo-statefoldinginmesophilic,thermophilic andhyperthermophiliccoldshockproteins,Nat.Struct.Biol.5(1998) 229–235,PMID:9501917.

[22]T.B.Topping,L.M.Gloss,Stabilityandfoldingmechanismofmesophilic,

thermophilicandhyperthermophilicarchaelhistones:theimportanceof

foldingintermediates,J.Mol.Biol.342(2004)247–260,http://dx.doi.org/10.

1016/j.jmb.2004.07.045.

[23]M.Wallgren,J.Aden,O.Pylypenko,T.Mikaelsson,L.B.Johansson,A.Rak,M.

Wolf-Watz,Extremetemperaturetoleranceofahyperthermophilicprotein

coupledtoresidualstructureintheunfoldedstate,J.Mol.Biol.379(2008)

845–858,http://dx.doi.org/10.1016/j.jmb.2008.04.007.

[24]K.A.Luke,C.L.Higgins,P.Wittung-Stafshede,Thermodynamicstabilityand

foldingofproteinsfromhyperthermophilicorganisms,FEBSJ.274(2007)

4023–4033,http://dx.doi.org/10.1111/j.1742-4658.2007.05955.x.

[25]A.Mukaiyama,K.Takano,Slowunfoldingofmonomericproteinsfrom

hyperthermophileswithreversibleunfolding,Int.J.Mol.Sci.10(2009)

1369–1385,http://dx.doi.org/10.3390/ijms10031369.

[26]K.Ogasahara,M.Nakamura,S.Nakura,S.Tsunasawa,I.Kato,T.Yoshimoto,K.

Yutani,Theunusuallyslowunfoldingratecausesthehighstabilityof

pyrrolidonecarboxylpeptidasefromahyperthermophile,Pyrococcusfuriosus:

equilibriumandkineticstudiesofguanidinehydrochloride-induced

unfoldingandrefolding,Biochemistry37(1998)17537–17544,http://dx.doi.

org/10.1021/bi9814585.

[27]J.Okada,T.Okamoto,A.Mukaiyama,T.Tadokoro,D.J.You,H.Chon,Y.Koga,K.

Takano,S.Kanaya,Evolutionandthermodynamicsoftheslowunfoldingof

hyperstablemonomericproteins,BMCEvol.Biol.10(2010)207,http://dx.doi.

org/10.1186/1471-2148-10-207.

[28]B.A.Kelch,D.A.Agard,Mesophileversusthermophile:insightsintothe

structuralmechanismsofkineticstability,J.Mol.Biol.370(2007)784–795,

http://dx.doi.org/10.1016/j.jmb.2007.04.078.

[29]P.Wittung-Stafshede,Slowunfoldingexplainshighstabilityofthermostable

ferredoxins:commonmechanismgoverningthermostability?Biochim.

Biophys.Acta2004(1700)1–4,http://dx.doi.org/10.1016/j.bbapap.2004.04.

002.

[30]X.Du,P.Sang,Y.L.Xia,Y.Li,J.Liang,S.M.Ai,X.L.Ji,Y.X.Fu,S.Q.Liu,

Comparativethermalunfoldingstudyofpsychrophilicandmesophilic

subtilisin-likeserineproteasesbymoleculardynamicssimulations,J.Biomol.

Struct.Dyn.35(2017)1500–1517,http://dx.doi.org/10.1080/07391102.2016.

1188155.

[31]A.Cipolla,S.D’Amico,R.Barumandzadeh,A.Matagne,G.Feller,Stepwise

adaptationstolowtemperatureasrevealedbymultiplemutantsof

psychrophilicalpha-amylasefromAntarcticbacterium,J.Biol.Chem.286

(2011)38348–38355,http://dx.doi.org/10.1074/jbc.M111.274423.

[37]F.U.Hartl,M.Hayer-Hartl,Convergingconceptsofproteinfoldinginvitroand

invivo,Nat.Struct.Mol.Biol.16(2009)574–581,http://dx.doi.org/10.1038/

nsmb.1591.

[38]A.T.Large,M.D.Goldberg,P.A.Lund,Chaperonesandproteinfoldinginthe

archaea,Biochem.Soc.Trans.37(2009)46–51,http://dx.doi.org/10.1042/

BST0370046.

[39]R.L.Baldwin,Thesearchforfoldingintermediatesandthemechanismof

proteinfolding,Annu.Rev.Biophys.37(2008)1–21,http://dx.doi.org/10.

1146/annurev.biophys.37.032807.125948.

[40]F.X.Schmid,Prolylisomerizationinproteinfolding,in:J.Buchner,T.

Kiefhaber(Eds.),ProteinFoldingHandbook,Wiley-VCH,Weinheim,2005,pp.

916–945,http://dx.doi.org/10.1002/9783527619498.ch25.

[41]G.Zoldak,T.Aumuller,C.Lucke,J.Hritz,C.Oostenbrink,G.Fischer,F.X.

Schmid,Alibraryoffluorescentpeptidesforexploringthesubstrate

specificitiesofprolylisomerases,Biochemistry48(2009)10423–10436,

http://dx.doi.org/10.1021/bi9014242.

[42]K.Watanabe,T.Masuda,H.Ohashi,H.Mihara,Y.Suzuki,Multipleproline

substitutionscumulativelythermostabilizeBacilluscereusATCC7064oligo-1,

6-glucosidase.Irrefragableproofsupportingtheprolinerule,Eur.J.Biochem.

226(1994)277–283,http://dx.doi.org/10.1111/j.1432-1033.1994.tb20051.x.

[43]C.Scholz,G.Stoller,T.Zarnt,G.Fischer,F.X.Schmid,Cooperationofenzymatic

andchaperonefunctionsoftriggerfactorinthecatalysisofproteinfolding,

EMBOJ.16(1997)54–58,http://dx.doi.org/10.1093/emboj/16.1.54.

[44]T.Maruyama,R.Suzuki,M.Furutani,Archaealpeptidylprolylcis-trans

isomerases(PPIases)update2004,Front.Biosci.9(2004)1680–1720,http://

dx.doi.org/10.2741/1361.

[45]S.D’Amico,J.C.Marx,C.Gerday,G.Feller,Activity-stabilityrelationshipsin

extremophilicenzymes,J.Biol.Chem.278(2003)7891–7896,http://dx.doi.

org/10.1074/jbc.M212508200.

[46]S.T.Ladani,M.G.Souffrant,A.Barman,D.Hamelberg,Computational

perspectiveandevaluationofplausiblecatalyticmechanismsof

peptidyl-prolylcis-transisomerases,Biochim.Biophys.Acta2015(1850)

1994–2004,http://dx.doi.org/10.1016/j.bbagen.2014.12.023.

[47]F.Piette,S.D’Amico,C.Struvay,G.Mazzucchelli,J.Renaut,M.L.Tutino,A.

Danchin,P.Leprince,G.Feller,Proteomicsoflifeatlowtemperatures:trigger

factoristheprimarychaperoneintheAntarcticbacteriumPseudoalteromonas

haloplanktisTAC125,Mol.Microbiol.76(2010)120–132,http://dx.doi.org/10.

1111/j.1365-2958.2010.07084.x.

[48]F.Piette,C.Struvay,G.Feller,Theproteinfoldingchallengeinpsychrophiles:

factsandcurrentissues,Environ.Microbiol.13(2011)1924–1933,http://dx.

doi.org/10.1111/j.1462-2920.2011.02436.x.

[49]G.Feller,D.d’Amico,C.Gerday,Thermodynamicstabilityofacold-active

alpha-amylasefromtheAntarcticbacteriumAlteromonashaloplanctis,

Biochemistry38(1999)4613–4619,http://dx.doi.org/10.1021/bi982650+.

[50]T.Thomas,R.Cavicchioli,Effectoftemperatureonstabilityandactivityof

elongationfactor2proteinsfromAntarcticandthermophilicmethanogens,J.

Bacteriol.182(2000)1328–1332,

http://dx.doi.org/10.1128/JB.182.5.1328-1332.2000.

[51]T.Collins,M.A.Meuwis,C.Gerday,G.Feller,Activity,stabilityandflexibilityin

glycosidasesadaptedtoextremethermalenvironments,J.Mol.Biol.328

(2003)419–428,http://dx.doi.org/10.1016/S0022-2836(03)00287-0.

[52]D.Georlette,B.Damien,V.Blaise,E.Depiereux,V.N.Uversky,C.Gerday,G.

Feller,Structuralandfunctionaladaptationstoextremetemperaturesin

psychrophilic,mesophilic,andthermophilicDNAligases,J.Biol.Chem.278

(2003)37015–37023,http://dx.doi.org/10.1074/jbc.M305142200.

[53]A.Cipolla,F.Delbrassine,J.L.DaLage,G.Feller,Temperatureadaptationsin

psychrophilic,mesophilicandthermophilicchloride-dependent

alpha-amylases,Biochimie94(2012)1943–1950,http://dx.doi.org/10.1016/j.

biochi.2012.05.013.

[54]D.M.LeMaster,J.Tang,G.Hernandez,Absenceofkineticthermalstabilization

inahyperthermophilerubredoxinindicatedby40microsecondfoldinginthe

presenceofirreversibledenaturation,Proteins57(2004)118–127,http://dx.

doi.org/10.1002/prot.20181.

[55]M.Sawano,H.Yamamoto,K.Ogasahara,S.Kidokoro,S.Katoh,T.Ohnuma,E.

Katoh,S.Yokoyama,K.Yutani,Thermodynamicbasisforthestabilitiesof

threeCutA1sfromPyrococcushorikoshii,Thermusthermophilus,andOryza

sativa,withunusuallyhighdenaturationtemperatures,Biochemistry47

[56]W.J.Becktel,J.A.Schellman,Proteinstabilitycurves,Biopolymers26(1987)

1859–1877,http://dx.doi.org/10.1002/bip.360261104.

[57]P.L.Privalov,Stabilityofproteins:smallglobularproteins,Adv.ProteinChem. 33(1979)167–241,PMID:44431.

[58]P.L.Privalov,Colddenaturationofproteins,Crit.Rev.Biochem.Mol.Biol.25

(1990)281–305,http://dx.doi.org/10.3109/10409239009090613.

[59]S.Kumar,R.Nussinov,Experiment-guidedthermodynamicsimulationson

reversibletwo-stateproteins:implicationsforproteinthermostability,

Biophys.Chem.111(2004)235–246,http://dx.doi.org/10.1016/j.bpc.2004.06.

005.

[60]A.Razvi,J.M.Scholtz,Lessonsinstabilityfromthermophilicproteins,Protein

Sci.15(2006)1569–1578,http://dx.doi.org/10.1110/ps.062130306.

[61]D.C.Rees,A.D.Robertson,Somethermodynamicimplicationsforthe

thermostabilityofproteins,ProteinSci.10(2001)1187–1194,http://dx.doi.

org/10.1110/ps.180101.

[62]S.Kumar,C.J.Tsai,R.Nussinov,Maximalstabilitiesofreversibletwo-state

proteins,Biochemistry41(2002)5359–5374,http://dx.doi.org/10.1021/

bi012154c.

[63]O.Garcia-Arribas,R.Mateo,M.M.Tomczak,P.L.Davies,M.G.Mateu,

Thermodynamicstabilityofacold-adaptedprotein,typeIIIantifreeze

protein,andenergeticcontributionofsaltbridges,ProteinSci.16(2007)

227–238,http://dx.doi.org/10.1110/ps.062448907.

[64]S.Knapp,A.Karshikoff,K.D.Berndt,P.Christova,B.Atanasov,R.Ladenstein,

ThermalunfoldingoftheDNA-bindingproteinSso7dfromthe

hyperthermophileSulfolobussolfataricus,J.Mol.Biol.264(1996)1132–1144,

http://dx.doi.org/10.1006/jmbi.1996.0701.

[65]B.S.McCrary,S.P.Edmondson,J.W.Shriver,Hyperthermophileproteinfolding

thermodynamics:differentialscanningcalorimetryandchemical

denaturationofSac7d,J.Mol.Biol.264(1996)784–805,http://dx.doi.org/10.

1006/jmbi.1996.0677.

[66]S.Robic,M.Guzman-Casado,J.M.Sanchez-Ruiz,S.Marqusee,Roleofresidual

structureintheunfoldedstateofathermophilicprotein,Proc.Natl.Acad.Sci.

U.S.A.100(2003)11345–11349,http://dx.doi.org/10.1073/pnas.

1635051100.

[67]P.L.Privalov,A.I.Dragan,Microcalorimetryofbiologicalmacromolecules,

Biophys.Chem.126(2007)16–24,http://dx.doi.org/10.1016/j.bpc.2006.05.

004.

[68]G.I.Makhatadze,P.L.Privalov,Energeticsofproteinstructure,Adv.Protein. Chem.47(1995)307–425,PMID:8561051.

[69]M.S.Urbieta,E.R.Donati,K.G.Chan,S.Shahar,L.L.Sin,K.M.Goh,Thermophiles

inthegenomicera:biodiversity,science,andapplications,Biotechnol.Adv.

33(2015)633–647,http://dx.doi.org/10.1016/j.biotechadv.2015.04.007.

[70]C.Moissl-Eichinger,C.Cockell,P.Rettberg,Venturingintonewrealms?

Microorganismsinspace,FEMSMicrobiol.Rev.40(2016)722–737,http://dx.